G2/M DNA DAMAGE CHECKPOINT IN BUDDING YEAST

芽殖酵母中的 G2/M DNA 损伤检查点

• 批准号: 2022428
• 负责人: TED A. WEINERT
• 金额: $ 19.87万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022428
• 关键词: DNA binding protein; DNA damage; DNA repair; Saccharomyces cerevisiae; cell cycle; fungal genetics; gene expression; gene mutation; immunofluorescence technique; protein purification; site directed mutagenesis; temperature sensitive mutant; yeast two hybrid system

DESCRIPTION: In eukaryotic cells, DNA damage causes arrest of the cell cycle to allow repair of this damage. A number of genes, termed "checkpoint" genes, required for this arrest have been identified in yeast and in other organisms. Human checkpoint genes include p53 and ATM and mutations in these genes are associated with increased probability of developing cancer. Dr. Weinert suggests that there may be three types of checkpoint proteins: 1) sensor proteins which bind and, perhaps, process DNA damage, 2) signal proteins that interact with the sensor protein and transduce a signal to target genes, and 3) the target genes, which directly mediate cell cycle arrest. His first series of experiments concern how sensor proteins act on DNA damage. Strains with the cdc13 mutation develop long single-stranded regions near the end of the chromosome. Dr. Weinert has found that mutations in the rad24 checkpoint gene reduced this degradation, and mutations in the rad9 checkpoint gene increased the degradation. He will determine whether the degradation occurs 3' to 5' or 5' to 3', and he will examine the effects of other mutations (for example, rad17) on the degradation process. The Rad17p shares homology with Rec1p, a known 3' to 5' exonuclease. Dr. Weinert will purify Rad17p and determine whether Rad17p is a 3' to 5' exonuclease. He will also try to localize Rad17p, Rad24 and Mec3p to sites of meiosis-specific DSB's using immunofluorescence. The point of these studies is to test the hypothesis that these proteins represent sensor proteins that process the cdc13-caused DNA damage. The second part of the proposal is designed to address two questions. First, is processing of DNA damage by Rad24p and Rad17p required to repair this DNA damage? Second, does cell cycle arrest require Rad24p and Rad17p to process the DNA damage? Dr. Weinert will try to answer these questions by mutating RAD17 and RAD24, and determining whether any of the mutant alleles show separation of function. For example, if he obtains a rad17 mutant that does not process DNA damage, but does show cell-cycle arrest, he will have demonstrated that DNA processing is not required for cell-cycle arrest. The third part of the proposal concerns new checkpoint mutants. His previous hunts involved looking for mutants that had poor viability after exposure to DNA damage. His new hunt will employ a direct search for cells that fail to show G2/M arrest in response to DNA damage. He hopes that this search will identify target genes of the checkpoint response. The last series of experiments investigate the order of function of genes in the checkpoint pathway. Dr. Weinert proposes looking at protein-protein interactions between different checkpoint proteins using the two-hybrid system or affinity columns. He also proposes two types of epistasis tests. Work from the Elledge lab suggests that the Rad53p is phosphorylated as part of the checkpoint response and functions downstream of Mec1p and the sensor proteins. Dr. Weinert will attempt to confirm this conclusion. If the conclusion is confirmed, he will determine the effect of various checkpoint mutations on the phosphorylation of Rad53p. He will also attempt to isolate a mec1 mutant with the phenotype of cold-sensitive constitutive function. He will then examine interactions with other mutants. A double mutant with a mec1 constitutive and a mutant in a gene that functions downstream of MEC1 should fail to show arrest at the restrictive temperature.

描述：在真核细胞中，DNA 损伤会导致细胞停滞 循环以修复此损坏。 许多基因，称为 酵母中已鉴定出这种逮捕所需的“检查点”基因 以及其他生物体中。 人类检查点基因包括 p53 和 ATM 这些基因的突变与增加的可能性有关 发展为癌症。 Weinert 博士认为可能存在三种类型 检查点蛋白：1) 结合并可能进行处理的传感器蛋白 DNA 损伤，2) 与传感器蛋白相互作用的信号蛋白和 将信号转导至目标基因，3) 目标基因，直接 介导细胞周期停滞。 他的第一个系列实验涉及传感器蛋白如何作用于 DNA 损害。 带有 cdc13 突变的菌株形成长单链 靠近染色体末端的区域。 韦纳特博士发现 rad24 检查点基因的突变减少了这种降解，并且 rad9 检查点基因的突变加剧了降解。 他会 确定降解是发生在 3' 到 5' 还是 5' 到 3'，他会 检查其他突变（例如 rad17）对 降解过程。 Rad17p 与 Rec1p 具有同源性，Rec1p 是已知的 3' 5'核酸外切酶。 Weinert博士将纯化Rad17p并确定Rad17p是否 是一种 3' 至 5' 核酸外切酶。 他还将尝试本地化 Rad17p、Rad24 和 使用免疫荧光将 Mec3p 连接至减数分裂特异性 DSB 位点。 这 这些研究的重点是检验这些蛋白质的假设 代表处理 cdc13 引起的 DNA 损伤的传感器蛋白。 该提案的第二部分旨在解决两个问题。 首先，修复所需的 Rad24p 和 Rad17p 对 DNA 损伤的处理是否需要 这种DNA损伤？ 二、细胞周期停滞需要Rad24p和Rad17p吗 处理DNA损伤？ Weinert博士将尝试回答这些问题 通过突变 RAD17 和 RAD24，并确定是否有任何突变体 等位基因显示功能分离。 例如，如果他获得了 rad17 突变体不处理DNA损伤，但确实表现出细胞周期停滞，他 将证明细胞周期不需要DNA加工 逮捕。 该提案的第三部分涉及新的检查点突变体。 他的 以前的狩猎涉及寻找在之后生存能力较差的突变体 暴露于 DNA 损伤。 他的新狩猎将采用直接寻找细胞的方法 未能显示出因 DNA 损伤而导致的 G2/M 期停滞。 他希望这 搜索将识别检查点反应的目标基因。 最后一系列实验研究了基因的功能顺序 检查点路径。 韦纳特博士建议研究蛋白质-蛋白质 使用双杂交方法观察不同检查点蛋白之间的相互作用 系统或亲和柱。 他还提出了两种类型的上位测试。 Elledge 实验室的工作表明 Rad53p 作为一部分被磷酸化 Mec1p 和传感器下游的检查点响应和功能 蛋白质。 韦纳特博士将尝试证实这一结论。 如果 结论确定后，他将确定各个检查点的效果 Rad53p 磷酸化突变。 他还将尝试隔离 具有冷敏感本构功能表型的 mec1 突变体。 然后他将检查与其他突变体的相互作用。 双突变体 mec1 组成型基因和在 MEC1 下游发挥作用的基因的突变体 在限制温度下不应表现出停滞。